title: Single-molecule spectroscopy of the unexpected collapse of an unfolded protein at low pH.
abstract: The dimensions of intrinsically disordered and unfolded proteins critically depend on the solution conditions, such as temperature, pH, ionic strength, and osmolyte or denarurant concentration. However, a quantitative understanding of how the complex combination of chain-chain and chain-solvent interactions is affected by the solvent is still missing. Here, we take a step towards this goal by investigating the combined effect of pH and denaturants on the dimensions of an unfolded protein. We use single-molecule fluorescence spectroscopy to extract the dimensions of unfolded cold shock protein (CspTm) in mixtures of the denaturants urea and guanidinium chloride (GdmCl) at neutral and acidic pH. Surprisingly, even though a change in pH from 7 to 2.9 increases the net charge of CspTm from -3.8 to +10.2, the radius of gyration of the chain is very similar under both conditions, indicating that protonation of acidic side chains at low pH results in additional hydrophobic interactions. We use a simple shared binding site model that describes the joint effect of urea and GdmCl, together with polyampholyte theory and an ion cloud model that includes the chemical free energy of counterion interactions and side chain protonation, to quantify this effect.
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Hagen
authors.initials: H
authors.lastname: Hofmann
authors.forename: Daniel
authors.initials: D
authors.lastname: Nettels
authors.forename: Benjamin
authors.initials: B
authors.lastname: Schuler
mesh_terms: D057773:Cold Shock Proteins and Peptides / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D058767:Protein Unfolding; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000737:chemistry
chemical_list: D057773:Cold Shock Proteins and Peptides; D014508:Urea; D019791:Guanidine